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1.  HIV shedding in the oral cavity: an assessment of HIV type, immunovirologic, demographic and oral factors 
Objective
To quantify the prevalence and burden of HIV type 2 (HIV-2) and HIV-1 RNA in the oral cavity of antiretroviral therapy-naive HIV-infected Senegalese individuals and to identify correlates of oral HIV viral loads.
Design
A cross-sectional study of 163 HIV-1 and 27 HIV-2-infected antiretroviral therapy-naive Senegalese adults.
Methods
Participants received clinical and oral exams and provided blood and oral wash samples for viral load and plasma CD4 count ascertainment. Logistic and interval regression models were used to identify univariate and multivariable associations between presence and level of oral HIV RNA and various immunovirologic, local and demographic factors.
Results
Presence of detectable oral HIV RNA was less common in HIV-2-infected compared with HIV-1-infected study participants (33% vs 67%, OR 0.25, 95% CI 0.11 to 0.59). HIV type was no longer associated with oral shedding of HIV when plasma viral load was considered. Detection of oral HIV RNA was associated with increased plasma viral load in both HIV-1-infected and HIV-2-infected individuals (HIV-1, OR 1.89, 95% CI 1.24 to 2.61; HIV-2, OR 1.93, 95% CI 1.1 to 3.39). Oral HIV-1 detection was also associated with periodontal disease (OR 3.02, 95% CI 1.16 to 7.87).
Conclusions
Oral shedding of HIV-2 RNA is less common than HIV-1 RNA, a likely consequence of lower overall viral burden. Both systemic and local factors may contribute to shedding of HIV in the oral cavity.
doi:10.1136/sextrans-2011-050231
PMCID: PMC4028315  PMID: 22250179
2.  QUANTITATIVE HUMAN PAPILLOMAVIRUS 16 AND 18 LEVELS IN INCIDENT INFECTIONS AND CERVICAL LESION DEVELOPMENT 
Journal of medical virology  2009;81(4):713-721.
Human papillomavirus (HPV) RNA levels may be a more sensitive early indicator of predisposition to carcinogenesis than DNA levels. We evaluated whether levels of HPV-16 and HPV-18 DNA and messenger RNA (mRNA) in newly detected infections are associated with cervical lesion development. Female university students were recruited from 1990-2004. Cervical samples for HPV DNA, HPV mRNA, and Papanicolaou testing were collected tri-annually, and women were referred for colposcopically-directed biopsy when indicated. Quantitative real-time polymerase chain reaction of L1 and E7 DNA and E7 mRNA was performed on samples from women with HPV-16 and HPV-18 infections that were incidently detected by consensus PCR. Adjusting for other HPV types, increasing E7 cervical HPV-16 mRNA levels at the time of incident HPV-16 DNA detection were associated with an increased risk of cervical intraepithelial neoplasia grade 2 to 3 (HR per 1 log10 increase in mRNA=6.36,95%CI=2.00-20.23). Increasing HPV-16 mRNA levels were also associated with an increased risk of cervical squamous intraepithelial lesions; the risk was highest at the incident positive visit and decreased over time. Neither HPV-16 E7 DNA levels nor HPV-18 E7 DNA nor mRNA levels were significantly associated with cervical lesion development. Report of >1 new partner in the past 8 months (relative to no new partners) was associated with increased HPV mRNA (viral level ratio [VLR]=10.05,95%CI=1.09-92.56) and increased HPV DNA (VLR=16.80,95%CI=1.46-193.01). In newly detected HPV-16 infections, increasing levels of E7 mRNA appear to be associated with an increased risk of developing cervical pre-cancer.
doi:10.1002/jmv.21450
PMCID: PMC3984467  PMID: 19235870
HPV; viral load; mRNA; cervical pre-cancer
3.  Persistence of Newly Detected Human Papillomavirus Type 31 Infection, Stratified by Variant Lineage 
Variants of human papillomavirus (HPV) type 31 have been shown to be related both to risk of cervical lesions and racial composition of a population. It is largely undetermined whether variants differ in their likelihood of persistence. Study subjects were women who participated in the ASCUS-LSIL Triage Study and who had a newly detected HPV31 infection during a 2-year follow-up with 6-month intervals. HPV31 isolates were characterized by sequencing and assigned to 1 of 3 variant lineages. Loss of the newly detected HPV31 infection was detected in 76 (47.5%) of the 160 women (32/67 with A variants, 16/27 with B variants, and 28/66 with C variants). The adjusted hazard ratio associating loss of the infection was 1.2 (95% CI, 0.7–2.1) for women with A variants and 2.1 (95% CI, 1.2–3.5) for women with B variants as compared to those with C variants. Infections with A and C variants were detected in 50 and 41 Caucasian women and in 15 and 23 African-American women, respectively. The likelihood of clearance of the infection was significantly lower in African-American women with C variants than in African-American women with A variants (P=0.05). There was no difference in the likelihood of clearance between A and C variants among Caucasian women. Our data indicated that infections with B variants were more likely to resolve than those with C variants. The difference in clearance of A versus C variants in African-Americans but not in Caucasians suggests a possibility of the race-related influence in retaining the variant-specific infection.
doi:10.1002/ijc.27689
PMCID: PMC3465533  PMID: 22729840
Human Papillomavirus; Variants; Persistence
4.  Lower levels of HIV-2 than HIV-1 in the female genital tract: correlates and longitudinal assessment of viral shedding 
AIDS (London, England)  2008;22(18):2517-2525.
Background
The differing magnitude of the HIV-1 and HIV-2 epidemics is likely a consequence of differing transmission rates between the two viruses. Similar to other sexually transmitted pathogens, risk of HIV-1 and HIV-2 transmission is likely associated with the presence and amount of HIV in the genital tract. Thus, understanding patterns of, and risk factors for HIV genital tract shedding is critical to effective control of HIV transmission.
Methods
We evaluated HIV DNA and RNA detection in cervicovaginal specimens among 168 HIV-1 and 50 HIV-2-infected women in Senegal, West Africa. In a subset of 31 women (20 with HIV-1, 11 with HIV-2), we conducted a prospective study in which cervicovaginal specimens were taken at 3-day intervals over a 6-week period.
Results
We found significantly lower rates and levels of HIV-2 RNA (58% shedding; 13% with >1000 copies/ml) in the female genital tract than HIV-1 RNA (78% shedding; 40% with >1000 copies/ml) (P =0.005 and 0.005, respectively), and shedding correlated with plasma viral load irrespective of virus type (odds ratio =1.9, 95% confidence interval =1.3–2.8 for each log10 increase in HIV viral RNA). Plasma viral load, not HIV type, was the strongest predictor of genital viral load. Over 80% of closely monitored women, regardless of HIV type, had at least intermittent HIV RNA detection during every 3-day sampling over a 6-week time period.
Conclusion
These data help in explaining the different transmission rates between HIV-1 and HIV-2 and may provide new insights regarding prevention.
doi:10.1097/QAD.0b013e328315cdbc
PMCID: PMC3726195  PMID: 19005275
Africa; cervix; women; HIV-1; HIV-2; Senegal; vagina; virus shedding
5.  Complex Patterns of Protease Inhibitor Resistance among Antiretroviral Treatment-Experienced HIV-2 Patients from Senegal: Implications for Second-Line Therapy 
Protease inhibitor (PI)-based antiretroviral therapy (ART) can effectively suppress HIV-2 plasma load and increase CD4 counts; however, not all PIs are equally active against HIV-2, and few data exist to support second-line therapy decisions. To identify therapeutic options for HIV-2 patients failing ART, we evaluated the frequency of PI resistance-associated amino acid changes in HIV-2 sequences from a cohort of 43 Senegalese individuals receiving unboosted indinavir (n = 18 subjects)-, lopinavir/ritonavir (n = 4)-, or indinavir and then lopinavir/ritonavir (n = 21)-containing ART. Common protease substitutions included V10I, V47A, I54M, V71I, I82F, I84V, L90M, and L99F, and most patients harbored viruses containing multiple changes. Based on genotypic data, we constructed a panel of 15 site-directed mutants of HIV-2ROD9 containing single- or multiple-treatment-associated amino acid changes in the protease-encoding region of pol. We then quantified the susceptibilities of the mutants to the HIV-2 “active” PIs saquinavir, lopinavir, and darunavir using a single-cycle assay. Relative to wild-type HIV-2, the V47A mutant was resistant to lopinavir (6.3-fold increase in the mean 50% effective concentration [EC50]), the I54M variant was resistant to darunavir and lopinavir (6.2- and 2.7-fold increases, respectively), and the L90M mutant was resistant to saquinavir (3.6-fold increase). In addition, the triple mutant that included I54M plus I84V plus L90M was resistant to all three PIs (31-, 10-, and 3.8-fold increases in the mean EC50 for darunavir, saquinavir, and lopinavir, respectively). Taken together, our data demonstrate that PI-treated HIV-2 patients frequently harbor viruses that exhibit complex patterns of PI cross-resistance. These findings suggest that sequential PI-based regimens for HIV-2 treatment may be ineffective.
doi:10.1128/AAC.00405-13
PMCID: PMC3716120  PMID: 23571535
6.  Prevalence and risk factors for oncogenic HPV infections in high-risk mid-adult women 
Sexually transmitted diseases  2012;39(11):848-856.
Background
The epidemiology of high-risk (hr) HPV infections in mid-adult women with new sex partners is undefined.
Methods
We analyzed baseline data from 518 25–65 year old female online daters. Women were mailed questionnaires and kits for self-collecting vaginal specimens for PCR-based hrHPV testing. Risk factors for infection were identified using Poisson regression models to obtain prevalence ratios (PRs).
Results
The prevalence of hrHPV infection was 35.9%. In multivariate analysis restricted to sexually active women, the likelihood of hrHPV infection was associated with abnormal Pap test history (PR=1.42, 95% CI:1.10–1.84), lifetime number of sex partners >14 (relative to 1–4; PR=2.13, 95% CI:1.13–4.02 for 15–24 partners and PR=1.91, 95% CI:1.00–3.64 for ≥25 partners), male partners with ≥1 concurrent partnership (PR=1.34, 95% CI:1.05–1.71) and male partners whom the subject met online (PR=1.39, 95% CI:1.08–1.79). Age was inversely associated with infection only in women who were sexually inactive (PR=0.67 per 5-year age difference, adjusted for Pap history and lifetime number of partners). Compared to sexually inactive women, the likelihood of infection increased with increasing risk level, (from low-risk to high-risk partners) (p<.0001 by trend test). In multivariate analysis, infection with multiple versus single hrHPV types was inversely associated with ever having been pregnant (PR=0.64, 95% CI:0.46–0.90) and recent consistent condom use (PR=0.56, 95% CI:0.32–0.97), and positively associated with genital wart history (PR=1.43, 95% CI:1.03–1.99).
Conclusions
Measures of both cumulative and recent sexual history were associated with prevalent hrHPV infection in this high-risk cohort of mid-adult women.
doi:10.1097/OLQ.0b013e3182641f1c
PMCID: PMC3476060  PMID: 23064533
HPV; human papilloma virus; mid-adult; prevalence; risk factors
7.  Validation for Clinical Use of a Novel HIV-2 Plasma RNA Viral Load Assay Using the Abbott m2000 Platform 
Background
Optimal care of persons infected with human immunodeficiency virus type 2 (HIV-2) requires an accurate assessment of HIV-2 plasma viral load (VL), but no clinically-approved quantitative HIV-2 RNA VL assay exists.
Objectives
To validate a novel quantitative HIV-2 RNA assay for clinical and research use.
Study Design
The Abbott m2000sp/rt platform was adapted for quantification of HIV-2 RNA in plasma. Amplification targeted a region of the long terminal repeat conserved in Group A and B HIV-2. Electron microscopy-counted-HIV-2 standards, the WHO/NIBSC HIV-2 International Standard and clinical specimens (N=162) were used to determine the precision, sensitivity, specificity, linear range, accuracy, and clinical performance of the assay.
Results
The quantitative linear range of the HIV-2 RNA assay was 10–1,000,000 copies/mL (R2 >0.99), with a limit of detection of 8 copies/mL (95% CI, 5–18 copies/ml). The assay did not cross-react with HIV-1, and quantification of HIV-2 RNA was not affected by the presence of >5 log10 HIV-1 RNA copies/mL. The total standard deviation (SD) and intra- and inter- run SD were 0.095, 0.093 and 0.162, respectively, at nominal inputs of 3.7, 1.7 and 1.0 log10 HIV-2 RNA copies/mL. The HIV-2 WHO/NIBSC International Standard (1000 IU) was shown to contain 152 RNA copies/mL (95% CI 141–163). Overall, HIV-2 RNA was quantified at ≥ 10 copies/mL from 86 (53%) clinical specimens (median, 2.24 log10copies/mL; range 10–16870), and nine specimens (6%) had HIV-2 RNA detected at <10 copies/mL.
Conclusions
We developed and validated a highly-sensitive HIV-2 VL assay that is suitable for clinical and research use.
doi:10.1016/j.jcv.2012.06.024
PMCID: PMC3444162  PMID: 22832059
HIV-2; PCR; Real-time; diagnostic; plasma viral loads
8.  Disclosure of Genital Human Papillomavirus Infection to Female Sex Partners by Young Men 
Sexually Transmitted Diseases  2012;39(8):583-587.
A survey was administered to male university students testing positive for high-risk human papillomavirus. Disclosure was more likely in men with fewer partners, in main partnerships, and in longer partnerships. Disclosure was associated with discussing the Pap test/HPV vaccine with female partners and not associated with a worsening relationship.
doi:10.1097/OLQ.0b013e318254c982
PMCID: PMC3398400  PMID: 22797688
human papillomavirus; male; disclosure; sexually transmitted infection; sexual partnerships
9.  Lower levels of HIV RNA in semen in HIV-2 compared with HIV-1 infection: implications for differences in transmission 
AIDS (London, England)  2006;20(6):895-900.
Background and objectives
HIV-2 infection, in comparison with HIV-1, is characterized by lower plasma viral loads, slower CD4 cell count decline, decreased AIDS-related mortality, and lower rates of mother-to-child and sexual transmission. To gain further insight into why HIV-1 is more readily transmitted as compared with HIV-2, we analyzed semen and plasma HIV RNA levels in HIV-1 and HIV-2-positive men from Senegal.
Design and methods
Twenty-two HIV-1 and 10 HIV-2-infected subjects from the University of Dakar donated semen and blood samples for this analysis. HIV-1 and HIV-2 viral loads in semen and plasma were quantified using type-specific polymerase chain reaction assays.
Results
The mean age of the subjects was 37 and 40 years; mean CD4 cell count was 222 and 276 cells/µl and the mean plasma viral load was 4.7 and 3.0 log10 copies/ml for HIV-1 and HIV-2, respectively (P = 0.002). HIV RNA was detected in semen in 21 of 22 (95%) of HIV-1 and seven of 10 (70%) of HIV-2-infected subjects; P = 0.07). However, the levels of HIV RNA present in semen were markedly different between those with HIV-1 and HIV-2, with a mean of 4.4 log10 copies/ml among those with HIV-1 and a mean of 2.6 log10 copies/ml among those with HIV-2 (P < 0.001). In multivariate analysis, plasma viral load and HIV type, but not CD4 cell count, were independently predictive of semen viral load (P = 0.03, 0.05, 0.48, respectively)
Conclusions
These data suggest that differences in semen viral load between HIV-1 and HIV-2 may be in part responsible for the markedly different transmission rates of these two viruses. In addition, risk of male genital tract shedding strongly correlates with plasma viral loads. Interventions that decrease viral load may help decrease transmission of both HIV-1 and HIV-2.
doi:10.1097/01.aids.0000218554.59531.80
PMCID: PMC3726185  PMID: 16549974
HIV-1; HIV-2; virus shedding; semen; male; Africa; Senegal
10.  Emergence of Multiclass Drug–Resistance in HIV-2 in Antiretroviral-Treated Individuals in Senegal: Implications for HIV-2 Treatment in Resouce-Limited West Africa 
Background
The efficacy of various antiretroviral (ARV) therapy regimens for human immunodeficiency virus type 2 (HIV-2) infection remains unclear. HIV-2 is intrinsically resistant to the nonnucleoside reverse-transcriptase inhibitors and to enfuvirtide and may also be less susceptible than HIV-1 to some protease inhibitors (PIs). However, the mutations in HIV-2 that confer ARV resistance are not well characterized.
Methods
Twenty-three patients were studied as part of an ongoing prospective longitudinal cohort study of ARV therapy for HIV-2 infection in Senegal. Patients were treated with nucleoside reverse-transcriptase inhibitor (NRTI)– and PI (indinavir)–based regimens. HIV-2 pol genes from these patients were genotyped, and the mutations predictive of resistance in HIV-2 were assessed. Correlates of ARV resistance were analyzed.
Results
Multiclass drug–resistance mutations (NRTI and PI) were detected in strains in 30% of patients; 52% had evidence of resistance to at least 1 ARV class. The reverse-transcriptase mutations M184V and K65R, which confer high-level resistance to lamivudine and emtricitabine in HIV-2, were found in strains from 43% and 9% of patients, respectively. The Q151M mutation, which confers multinucleoside resistance in HIV-2, emerged in strains from 9% of patients. HIV-1–associated thymidine analogue mutations (M41L, D67N, K70R, L210W, and T215Y/F) were not observed, with the exception of K70R, which was present together with K65R and Q151M in a strain from 1 patient. Eight patients had HIV-2 with PI mutations associated with indinavir resistance, including K7R, I54M, V62A, I82F, L90M, L99F; 4 patients had strains with multiple PI resistance–associated mutations. The duration of ARV therapy was positively associated with the development of drug resistance (P = .02). Nine (82%) of 11 patients with HIV-2 with detectable ARV resistance had undetectable plasma HIV-2 RNA loads (<1.4 log10 copies/mL), compared with 3 (25%) of 12 patients with HIV-2 with detectable ARV resistance (P = .009). Patients with ARV-resistant virus had higher plasma HIV-2 RNA loads, compared with those with non–ARV-resistant virus (median, 1.7 log10 copies/mL [range, <1.4 to 2.6 log10 copies/mL] vs. <1.4 log10 copies/mL [range, <1.4 to 1.6 log10 copies/mL]; P = .003).
Conclusions
HIV-2–infected individuals treated with ARV therapy in Senegal commonly have HIV-2 mutations consistent with multiclass drug resistance. Additional clinical studies are required to improve the efficacy of primary and salvage treatment regimens for treating HIV-2 infection.
doi:10.1086/596504
PMCID: PMC3671065  PMID: 19143530
11.  Phenotypic susceptibility of HIV-2 to raltegravir: integrase mutations Q148R and N155H confer raltegravir resistance 
AIDS (London, England)  2011;25(18):2235-2241.
Objectives
Raltegravir is the first integrase strand transfer inhibitor approved for treating HIV-1 infection. Although emerging data suggest that raltegravir may also be useful for HIV-2 treatment, studies addressing the in-vitro susceptibility of HIV-2 to raltegravir are scarce, and the genetic pathways leading to raltegravir resistance in HIV-2 have not been adequately characterized. Our objectives were to directly compare the susceptibilities of HIV-1 and HIV-2 to raltegravir and to examine the role of mutations in HIV-2 integrase in emergent raltegravir resistance.
Materials and methods
Single-cycle and spreading infection assays were used to quantify the sensitivities of wild-type HIV-1 and HIV-2 strains to raltegravir. HIV-2 integrase mutants were constructed by site-directed mutagenesis, and the replication capacities and raltegravir susceptibilities of the resultant variants were analyzed in single-cycle assays.
Results
Raltegravir showed comparable activity against wild-type HIV-1 and HIV-2 in both single-cycle and spreading infections, with EC50 values in the low nanomolar range. Amino acid changes Q148R and N155H individually conferred resistance to raltegravir (14-fold and seven-fold, respectively), whereas the Y143C replacement had no statistically significant effect on raltegravir sensitivity. The combination of Q148R with N155H resulted in high-level raltegravir resistance (>1000-fold). In addition, all HIV-2 integrase variants tested showed impairments in replication capacity.
Conclusion
Our data support clinical studies of raltegravir for treating HIV-2 infection and show that the Q148R and N155H changes alone are sufficient for raltegravir resistance in HIV-2. Further efforts are needed to improve access to HIV-2-active antiretrovirals, including raltegravir, in resource-limited areas where HIV-2 is endemic.
doi:10.1097/QAD.0b013e32834d8e52
PMCID: PMC3652615  PMID: 21971360
HIV-1; HIV-2; integrase; N155H; Q148R; raltegravir; resistance; Y143C
12.  MicroRNA expression in ovarian carcinoma and its correlation with clinicopathological features 
Background
MicroRNA (miRNA) expression is known to be deregulated in ovarian carcinomas. However, limited data is available about the miRNA expression pattern for the benign or borderline ovarian tumors as well as differential miRNA expression pattern associated with histological types, grades or clinical stages in ovarian carcinomas. We defined patterns of microRNA expression in tissues from normal, benign, borderline, and malignant ovarian tumors and explored the relationship between frequently deregulated miRNAs and clinicopathologic findings, response to therapy, survival, and association with Her-2/neu status in ovarian carcinomas.
Methods
We measured the expression of nine miRNAs (miR-181d, miR-30a-3p, miR-30c, miR-30d, miR-30e-3p, miR-368, miR-370, miR-493-5p, miR-532-5p) in 171 formalin-fixed, paraffin-embedded ovarian tissue blocks as well as six normal human ovarian surface epithelial (HOSE) cell lines using Taqman-based real-time PCR assays. Her-2/neu overexpression was assessed in ovarian carcinomas (n = 109 cases) by immunohistochemistry analysis.
Results
Expression of four miRNAs (miR-30c, miR-30d, miR-30e-3p, miR-370) was significantly different between carcinomas and benign ovarian tissues as well as between carcinoma and borderline tissues. An additional three miRNAs (miR-181d, miR-30a-3p, miR-532-5p) were significantly different between borderline and carcinoma tissues. Expression of miR-532-5p was significantly lower in borderline than in benign tissues. Among ovarian carcinomas, expression of four miRNAs (miR-30a-3p, miR-30c, miR-30d, miR-30e-3p) was lowest in mucinous and highest in clear cell samples. Expression of miR-30a-3p was higher in well-differentiated compared to poorly differentiated tumors (P = 0.02), and expression of miR-370 was higher in stage I/II compared to stage III/IV samples (P = 0.03). In multivariate analyses, higher expression of miR-181d, miR-30c, miR-30d, and miR-30e-3p was associated with significantly better disease-free or overall survival. Finally, lower expression of miR-30c, miR-30d, miR-30e-3p and miR-532-5p was significantly associated with overexpression of Her-2/neu.
Conclusions
Aberrant expression of miRNAs is common in ovarian tumor suggesting involvement of miRNA in ovarian tumorigenesis. They are associated with histology, clinical stage, survival and oncogene expression in ovarian carcinoma.
doi:10.1186/1477-7819-10-174
PMCID: PMC3449188  PMID: 22925189
miRNA; Ovarian tumor; Her2/neu; Survival
13.  Broad and Potent Neutralizing Antibody Responses Elicited in Natural HIV-2 Infection 
Journal of Virology  2012;86(2):947-960.
Compared with human immunodeficiency virus type 1 (HIV-1), little is known about the susceptibility of HIV-2 to antibody neutralization. We characterized the potency and breadth of neutralizing antibody (NAb) responses in 64 subjects chronically infected with HIV-2 against three primary HIV-2 strains: HIV-27312A, HIV-2ST, and HIV-2UC1. Surprisingly, we observed in a single-cycle JC53bl-13/TZM-bl virus entry assay median reciprocal 50% inhibitory concentration (IC50) NAb titers of 1.7 × 105, 2.8 × 104, and 3.3 × 104, respectively. A subset of 5 patient plasma samples tested against a larger panel of 17 HIV-2 strains where the extracellular gp160 domain was substituted into the HIV-27312A proviral backbone showed potent neutralization of all but 4 viruses. The specificity of antibody neutralization was confirmed using IgG purified from patient plasma, HIV-2 Envs cloned by single-genome amplification, viruses grown in human CD4+ T cells and tested for neutralization sensitivity on human CD4+ T target cells, and, as negative controls, env-minus viruses pseudotyped with HIV-1, vesicular stomatitis virus, or murine leukemia virus Env glycoproteins. Human monoclonal antibodies (MAbs) specific for HIV-2 V3 (6.10F), V4 (1.7A), CD4 binding site (CD4bs; 6.10B), CD4 induced (CD4i; 1.4H), and membrane-proximal external region (MPER; 4E10) epitopes potently neutralized the majority of 32 HIV-2 strains bearing Envs from 13 subjects. Patient antibodies competed with V3, V4, and CD4bs MAbs for binding to monomeric HIV-2 gp120 at titers that correlated significantly with NAb titers. HIV-2 MPER antibodies did not contribute to neutralization breadth or potency. These findings indicate that HIV-2 Env is highly immunogenic in natural infection, that high-titer broadly neutralizing antibodies are commonly elicited, and that unlike HIV-1, native HIV-2 Env trimers expose multiple broadly cross-reactive epitopes readily accessible to NAbs.
doi:10.1128/JVI.06155-11
PMCID: PMC3255805  PMID: 22031948
14.  Viral Load in the Natural History of Human Papillomavirus Type 16 Infection: A Nested Case–control Study 
The Journal of Infectious Diseases  2011;203(10):1425-1433.
Background. Viral load may influence the course of human papillomavirus type 16 (HPV-16) infection.
Methods. This case-control study was nested within the 2-year Atypical Squamous Cells of Undetermined Significance and Low-Grade Squamous Intraepithelial Lesion Triage Study, in which women were followed semiannually for HPV and cervical intraepithelial neoplasia (CIN). Case patients (n = 62) were women diagnosed with CIN3 following HPV-16–positive detection at a follow-up visit. HPV-16–positive controls (n = 152) without CIN2 or CIN3 were matched to cases based on the follow-up visit in which viral load was measured. Real-time polymerase chain reaction was used for HPV-16 DNA quantification.
Results. The risk of CIN3 increased with increasing HPV-16 DNA load at the follow-up visit (odds ratio, 1.63; 95% confidence interval, 1.33–1.99 per 1 log10 unit increase); the association was not affected by whether HPV-16 was present at enrollment. When HPV-16 was present at both enrollment and follow-up, viral load remained high among cases (P = .77) but decreased substantially among controls (P = .004). Among women with HPV-16 found initially during follow-up, viral load in the first HPV-16–positive sample was associated with short-term persistence; load was higher in those with infection, compared with those without infection, 1 visit after the initial positivity (P = .001).
Conclusions. Viral load of newly detected infections and changes in viral load predict persistence and progression of HPV-16 infections.
doi:10.1093/infdis/jir049
PMCID: PMC3080901  PMID: 21415020
15.  Analysis of Tp53 Codon 72 Polymorphisms, Tp53 Mutations, and HPV Infection in Cutaneous Squamous Cell Carcinomas 
PLoS ONE  2012;7(4):e34422.
Background
Non-melanoma skin cancers are one of the most common human malignancies accounting for 2–3% of tumors in the US and represent a significant health burden. Epidemiology studies have implicated Tp53 mutations triggered by UV exposure, and human papilloma virus (HPV) infection to be significant causes of non-melanoma skin cancer. However, the relationship between Tp53 and cutaneous HPV infection is not well understood in skin cancers. In this study we assessed the association of HPV infection and Tp53 polymorphisms and mutations in lesional specimens with squamous cell carcinomas.
Methods
We studied 55 cases of histologically confirmed cutaneous squamous cell carcinoma and 41 controls for the presence of HPV infection and Tp53 genotype (mutations and polymorphism).
Results
We found an increased number of Tp53 mutations in the squamous cell carcinoma samples compared with perilesional or control samples. There was increased frequency of homozygous Tp53-72R polymorphism in cases with squamous cell carcinomas, while the Tp53-72P allele (Tp53-72R/P and Tp53-72P/P) was more frequent in normal control samples. Carcinoma samples positive for HPV showed a decreased frequency of Tp53 mutations compared to those without HPV infection. In addition, carcinoma samples with a Tp53-72P allele showed an increased incidence of Tp53 mutations in comparison carcinomas samples homozygous for Tp53-72R.
Conclusions
These studies suggest there are two separate pathways (HPV infection and Tp53 mutation) leading to cutaneous squamous cell carcinomas stratified by the Tp53 codon-72 polymorphism. The presence of a Tp53-72P allele is protective against cutaneous squamous cell carcinoma, and carcinoma specimens with Tp53-72P are more likely to have Tp53 mutations. In contrast Tp53-72R is a significant risk factor for cutaneous squamous cell carcinoma and is frequently associated with HPV infection instead of Tp53 mutations. Heterozygosity for Tp53-72R/P is protective against squamous cell carcinomas, possibly reflecting a requirement for both HPV infection and Tp53 mutations.
doi:10.1371/journal.pone.0034422
PMCID: PMC3335843  PMID: 22545084
16.  EARLY NATURAL HISTORY OF INCIDENT TYPE-SPECIFIC HUMAN PAPILLOMAVIRUS INFECTIONS IN NEWLY SEXUALLY ACTIVE YOUNG WOMEN 
BACKGROUND
Characterizing short-term detection patterns of young women’s incident alpha-genus human papillomavirus (HPV) infections may further understanding of HPV transmission.
METHODS
Between 2000–2007, we followed 18–22 year old female university students with triannual HPV DNA and Papanicolau testing. Using Kaplan-Meier methods, we estimated: duration of detectable, type-specific incident infections; time to re-detection (among infections that became undetectable); and time to cervical lesion development after incident infection. We evaluated risk factors for short-term persistent versus transient infection with logistic regression.
RESULTS
303 incident type-specific infections were detected in 85 sexually active women. Median time to first negative test after incident infection was 9.4 (95%CI:7.8–11.2) months; 90.6% of infections became undetectable within two years. 19.4% of infections that became undetectable were re-detected within one year. Cervical lesions were common, and 60% were positive for multiple HPV types in concurrent cervical swabs. Incident HPV detection in the cervix only (versus the vulva/vagina only or both sites) was associated with short-term transience.
CONCLUSIONS
While most incident infections became undetectable within two years, re-detection was not uncommon. Cervical lesions were a common early manifestation of HPV infection.
IMPACT
It remains unclear whether potentially modifiable risk factors can be identified to reduce infection duration (and transmission likelihood).
doi:10.1158/1055-9965.EPI-10-1108
PMCID: PMC3078690  PMID: 21173170
human papillomavirus; incidence; duration; persistence; women; epidemiology
17.  Detection of Human Papillomavirus DNA in Cutaneous Squamous Cell Carcinoma among Immunocompetent Individuals 
The presence of certain types of human papillomavirus (HPV) is a known risk factor for the development of anogenital squamous cell carcinomas (SCCs). A similar association has been hypothesized for cutaneous SCCs, although, to our knowledge, no studies to date have combined sensitive HPV DNA detection techniques with epidemiologic data controlling for known risk factors to explore the association. We designed a case–control study examining HPV prevalence using highly sensitive PCR-detection assays in tissue samples from 85 immunocompetent patients with histologically confirmed SCCs and 95 age-matched individuals without a prior history of skin cancer. A standardized interview was administered to all study subjects to collect information pertaining to potential confounding variables. The overall detection rate of HPV DNA was high in case lesions (54%) and perilesions (50%) and in both sun-exposed normal tissue (59%) and non-sun-exposed normal tissue (49%) from controls. In comparing case tissue to control tissue, there was no differential detection of HPV DNA across various HPV species. However, HPV DNA from β-papillomavirus species 2 was more likely to be identified in tumors than in adjacent healthy tissue among cases (paired analysis, odds ratio = 4.0, confidence interval = 1.3–12.0). The high prevalence of HPV DNA detected among controls suggests that HPV DNA is widely distributed among the general population. However, the differential detection of HPV β-papillomavirus species in tumors among cases suggests that certain HPV types may be involved in the progression of cutaneous SCCs.
doi:10.1038/sj.jid.5701227
PMCID: PMC3268673  PMID: 18185530
18.  Expression of Mir-21 and Mir-143 in Cervical Specimens Ranging from Histologically Normal through to Invasive Cervical Cancer 
PLoS ONE  2011;6(12):e28423.
Background
MicroRNA expression is severely disrupted in carcinogenesis, however limited evidence is available validating results from cell-line models in human clinical cancer specimens. MicroRNA-21 (mir-21) and microRNA-143 (mir-143) have previously been identified as significantly deregulated in a range of cancers including cervical cancer. Our goal was to investigate the expression patterns of several well-studied microRNA species in cervical samples and compare the results to cell line samples.
Methodology/Principal Findings
We measured the expression of mir-21 and mir-143 in 142 formalin-fixed, paraffin embedded (FFPE) cervical biopsy tissue blocks, collected from Dantec Oncology Clinic, Dakar, Senegal. MicroRNA expression analysis was performed using Taqman-based real-time PCR assays. Protein immunohistochemical staining was also performed to investigate target protein expression on 72 samples. We found that mir-21 expression increased with worsening clinical diagnosis but that mir-143 was not correlated with histology. These observations were in stark contrast to previous reports involving cervical cancer cell lines in which mir-143 was consistently down-regulated but mir-21 largely unaffected. We also identified, for the first time, that cytoplasmic expression of Programmed Cell Death Protein 4 PDCD4; a known target of mir-21) was significantly lower in women with invasive cervical carcinoma (ICC) in comparison to those with cervical intraepithelial neoplasia (2–3) or carcinoma in situ (CIN2-3/CIS), although there was no significant correlation between mir-21 and PDCD4 expression, despite previous studies identifying PDCD4 transcript as a known mir-21 target.
Conclusions
Whilst microRNA biomarkers have a number of promising features, more studies on expression levels in histologically defined clinical specimens are required to investigate clinical relevance of discovery-based studies. Mir-21 may be of some utility in predictive screening, given that we observed a significant correlation between mir-21 expression level and worsening histological diagnosis of cervical cancer.
doi:10.1371/journal.pone.0028423
PMCID: PMC3237431  PMID: 22194833
19.  Human Papillomavirus Type 16 Variants in Paired Enrollment and Follow-up Cervical Samples: Implications for a Proper Understanding of Type-Specific Persistent Infections 
The Journal of Infectious Diseases  2010;202(11):1667-1670.
Prospective studies of the persistence of human papilloma-virus (HPV) variants are rare and typically small. We sequenced HPV-16 variants in longitudinal pairs of specimens from 86 women enrolled in the ASCUS-LSIL Triage Study. A change of variants was identified in 4 women (4.7% [95% confidence interval, 1.3%-11.5%]). Among women with intervening HPV results (n = 60), a variant switch occurred in 2 of 11 who had evidence of intervening negativity for HPV-16, compared with 1 of 49 who consistently tested positive (P = .11). These results suggest the possibility that rare misclassification of transient infections as persistent infections occurs in natural history studies of type-specific HPV infections.
doi:10.1086/657083
PMCID: PMC3107552  PMID: 20977339
20.  Inverse Association between Methylation of Human Papillomavirus Type 16 DNA and Risk of Cervical Intraepithelial Neoplasia Grades 2 or 3 
PLoS ONE  2011;6(8):e23897.
Background
The clinical relevance of human papillomavirus type 16 (HPV16) DNA methylation has not been well documented, although its role in modulation of viral transcription is recognized.
Methods
Study subjects were 211 women attending Planned Parenthood clinics in Western Washington for routine Papanicolaou screening who were HPV16 positive at the screening and/or subsequent colposcopy visit. Methylation of 11 CpG dinucleotides in the 3′ end of the long control region of the HPV16 genome was examined by sequencing the cloned polymerase chain reaction products. The association between risk of CIN2/3 and degree of CpG methylation was estimated using a logistic regression model.
Results
CIN2/3 was histologically confirmed in 94 (44.5%) of 211 HPV16 positive women. The likelihood of being diagnosed as CIN2/3 increased significantly with decreasing numbers of methylated CpGs (meCpGs) in the 3′ end of the long control region (Pfor trend = 0.003). After adjusting for HPV16 variants, number of HPV16-positive visits, current smoking status and lifetime number of male sex partners, the odds ratio for the association of CIN2/3 with ≥4 meCpGs was 0.31 (95% confidence interval, 0.12–0.79). The proportion of ≥4 meCpGs decreased appreciably as the severity of the cervical lesion increased (Pfor trend = 0.001). The inverse association remained similar when CIN3 was used as the clinical endpoint. Although not statistically significant, the ≥4 meCpGs-related risk reduction was more substantial among current, as compared to noncurrent, smokers.
Conclusion
Results suggest that degree of the viral genome methylation is related to the outcome of an HPV16 cervical infection.
doi:10.1371/journal.pone.0023897
PMCID: PMC3161083  PMID: 21887341
21.  DNA hypermethylation of tumors from non-small cell lung cancer (NSCLC) patients is associated with gender and histologic type 
Background
We previously identified a number of genes which were methylated significantly more frequently in the tumor compared to the non-cancerous lung tissues from non-small cell lung cancer (NSCLC) patients. Detection of methylation profiles of genes in NSCLC could provide insight into differential pathways to malignancy and lead to strategies for better treatment of individuals with NSCLC.
Methods
We determined the DNA methylation status of 27 genes using quantitative MethyLight assays in lung tumor samples from 117 clinically well-characterized NSCLC patients.
Results
Hypermethylation was detected in one of more of the genes in 106 (91%) of 117 cases and was detected at high levels (Percentage of Methylation Reference (PMR)≥4%) in 79% of NSCLC cases. Methylation of APC, CCND2, KCNH5 and, RUNX was significantly more frequent in adenocarcinomas compared to squamous cell carcinomas (SCC), while methylation of CDKN2A was more common in SCC. Hypermethylation of KCNH5, KCNH8, and RARB was more frequent in females compared to males. Hypermethylation of APC and CCND2 was inversely associated with proliferation score assessed by Ki-67 level.
Conclusions
Our findings of differential gene hypermethylation frequencies in tumor tissues from patients with adenocarcinoma or squamous cell cancers and in females compared to males suggests that further investigation is warranted in order to more fully understand the potential disparate pathways and/or risk factors for NSCLC associated with histologic type and gender.
doi:10.1016/j.lungcan.2009.11.002
PMCID: PMC2888601  PMID: 19945765
hypermethylation; lung cancer; gender; histology
22.  HIV-2 Integrase Variation in Integrase Inhibitor-Naïve Adults in Senegal, West Africa 
PLoS ONE  2011;6(7):e22204.
Background
Antiretroviral therapy for HIV-2 infection is hampered by intrinsic resistance to many of the drugs used to treat HIV-1. Limited studies suggest that the integrase inhibitors (INIs) raltegravir and elvitegravir have potent activity against HIV-2 in culture and in infected patients. There is a paucity of data on genotypic variation in HIV-2 integrase that might confer intrinsic or transmitted INI resistance.
Methods
We PCR amplified and analyzed 122 HIV-2 integrase consensus sequences from 39 HIV-2–infected, INI-naive adults in Senegal, West Africa. We assessed genetic variation and canonical mutations known to confer INI-resistance in HIV-1.
Results
No amino acid-altering mutations were detected at sites known to be pivotal for INI resistance in HIV-1 (integrase positions 143, 148 and 155). Polymorphisms at several other HIV-1 INI resistance-associated sites were detected at positions 72, 95, 125, 154, 165, 201, 203, and 263 of the HIV-2 integrase protein.
Conclusion
Emerging genotypic and phenotypic data suggest that HIV-2 is susceptible to the new class of HIV integrase inhibitors. We hypothesize that intrinsic HIV-2 integrase variation at “secondary” HIV-1 INI-resistance sites may affect the genetic barrier to HIV-2 INI resistance. Further studies will be needed to assess INI efficacy as part of combination antiretroviral therapy in HIV-2–infected patients.
doi:10.1371/journal.pone.0022204
PMCID: PMC3134476  PMID: 21765953
23.  Accuracy and Cost-Effectiveness of Cervical Cancer Screening by High-Risk HPV DNA Testing of Self-Collected Vaginal Samples 
Objective
Estimate the accuracy and cost-effectiveness of cervical cancer screening strategies based on high-risk HPV DNA testing of self-collected vaginal samples.
Materials and Methods
A subset of 1,665 women (18-50 years of age) participating in a cervical cancer screening study were screened by liquid-based cytology and by high-risk HPV DNA testing of both self-collected vaginal swab samples and clinician-collected cervical samples. Women with positive/abnormal screening test results and a subset of women with negative screening test results were triaged to colposcopy. Based on individual and combined test results, five screening strategies were defined. Estimates of sensitivity and specificity for cervical intraepithelial neoplasia grade 2 or worse were calculated and a Markov model was used to estimate the incremental cost-effectiveness ratios (ICERs) for each strategy.
Results
Compared to cytology-based screening, high-risk HPV DNA testing of self-collected vaginal samples was more sensitive (68%, 95%CI=58%-78% versus 85%, 95%CI=76%-94%) but less specific (89%, 95%CI=86%-91% versus 73%, 95%CI=67%-79%). A strategy of high-risk HPV DNA testing of self-collected vaginal samples followed by cytology triage of HPV positive women, was comparably sensitive (75%, 95%CI=64%-86%) and specific (88%, 95%CI=85%-92%) to cytology-based screening. In-home self-collection for high-risk HPV DNA detection followed by in-clinic cytology triage had a slightly lower lifetime cost and a slightly higher quality-adjusted life expectancy than did cytology-based screening (ICER of triennial screening compared to no screening was $9,871/QALY and $12,878/QALY, respectively).
Conclusions
Triennial screening by high-risk HPV DNA testing of in-home, self-collected vaginal samples followed by in-clinic cytology triage was cost-effective.
doi:10.1097/LGT.0b013e3181cd6d36
PMCID: PMC2898894  PMID: 20592553
cervical cancer; screening; hpv; self-collect; cost-effectiveness
24.  DETECTION OF GENITAL HPV TYPES IN FINGERTIP SAMPLES FROM NEWLY SEXUALLY ACTIVE FEMALE UNIVERSITY STUDENTS 
Background
Little is known about detection of genital human papillomavirus (HPV) types in women’s fingertips. The study objectives were to determine the presence of genital HPV types in fingertip samples and agreement between fingertip and genital samples for detecting HPV.
Methods
At tri-annual visits, genital and fingertip samples were collected from female university students and tested for 37 HPV genotypes by PCR-based assay. Type-specific concordance between paired fingertip and genital samples was evaluated using a kappa statistic for percent positive agreement (“kappa +”). Paired samples with type-specific concordant fingertip and genital results were selected for variant characterization.
Results
A total of 357 fingertip samples were collected from 128 women. HPV prevalence in fingertip samples was 14.3%. Although percent positive agreement between fingertips and genitals for detecting type-specific HPV was low (17.8%; kappa+=0.17, 95%CI:0.10–0.25), 60.4% of type-specific HPV detected in the fingertips was detected in a concurrent genital sample. All but one of 28 paired concordant samples were positive for the same type-specific variant in the fingertip and genital sample. Re-detection of HPV types at the subsequent visit was more common in genital samples (73.3%) than in fingertip samples (14.5%) (p<.001).
Conclusions
Detection of genital HPV types in the fingertips was not uncommon. While impossible to distinguish between deposition of DNA from the genitals to the fingertips and true fingertip infection, the rarity of repeat detection in the fingertips suggests that deposition is more common.
Impact
Finger-genital transmission is plausible, but unlikely to be a significant source of genital HPV infection.
doi:10.1158/1055-9965.EPI-10-0226
PMCID: PMC2901391  PMID: 20570905
human papillomavirus; fingertip; genital; women; epidemiology
25.  Hypermethylation of CCND2 May Reflect a Smoking-Induced Precancerous Change in the Lung 
Journal of Oncology  2011;2011:950140.
It remains unknown whether tobacco smoke induces DNA hypermethylation as an early event in carcinogenesis or as a late event, specific to overt cancer tissue. Using MethyLight assays, we analyzed 316 lung tissue samples from 151 cancer-free subjects (121 ever-smokers and 30 never-smokers) for hypermethylation of 19 genes previously observed to be hypermethylated in nonsmall cell lung cancers. Only APC (39%), CCND2 (21%), CDH1 (7%), and RARB (4%) were hypermethylated in >2% of these cancer-free subjects. CCND2 was hypermethylated more frequently in ever-smokers (26%) than in never-smokers (3%). CCND2 hypermethylation was also associated with increased age and upper lobe sample location. APC was frequently hypermethylated in both ever-smokers (41%) and never-smokers (30%). BVES, CDH13, CDKN2A (p16), CDKN2B, DAPK1, IGFBP3, IGSF4, KCNH5, KCNH8, MGMT, OPCML, PCSK6, RASSF1, RUNX, and TMS1 were rarely hypermethylated (<2%) in all subjects. Hypermethylation of CCND2 may reflect a smoking-induced precancerous change in the lung.
doi:10.1155/2011/950140
PMCID: PMC3090638  PMID: 21577262

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